In cancer an increasing number of proteins involved in cell growth, including growth factors, receptors, intracellular mediators and transcription factors have been found to be altered through multiple mechanisms of activation. Dysregulation of human epidermal growth factor receptor (EGFR) family by over-expression or constitutive activation promotes tumor processes including angiogenesis and metastasis and is associated with poor prognosis in many human malignancies (Yarden Y, 2001; Mitsudomi T and Yatabe Y, 2010). The EGFR/ErbB family of receptor tyrosine kinases (RTK) comprises the four members: EGFR (also known as HER1 or ErbB1), ErbB2 (Neu, HER2), ErbB3 (HER3) and ErbB4 (HER4), which are type I transmembrane glycoproteins containing an extracellular ligand binding region, a single membrane-spanning region and an intracellular tyrosine-kinase-containing domain. Unlike the rest of the ErbB family, ErbB3 lacks tyrosine kinase activity and ErbB2 has no known ligand. EGF and transforming growth factor α bind directly only to EGFR, whereas neuregulins (also known as heregulins) are specific for ErbB3 and ErbB4 (Hynes N E and Lane H A, 2005). Ligand-induced activation of EGFR by dimerization mediates the activation of multiple downstream signaling pathways e.g the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/AKT, and signal transducers and activators of transcription 3 (STAT 3), which play pivotal roles in cellular events such as proliferation and survival (Schlessinger, 2004; Lurje and Lenz, 2009).
Two major classes of EGFR inhibitors are in clinical use: the anti-ErbB monoclonal antibodies, that bind to the extracellular domain of EGFR on the surface of tumour cells and small-molecule tyrosine-kinase inhibitors (TKI) that compete with ATP for binding to the tyrosine-kinase domain of the receptor (Li et al., 2005; Spicer J and Harper P, 2005; de La Motte Rouge et al., 2007; Cardó-Vilaa et al., 2010). The treatment of tumour cells with these agents affects many of the intracellular pathways that are essential for cancer development and progression. However, despite clinical success, the vast majority of patients receiving these treatments show primary or acquired resistance to the inhibitors (Kruser T J and Wheeler D L, 2010). Thus, new strategies to overcome TKI resistance are under active exploration and there is the urgent need to design new EGFR-targeting drugs for a more specific and selective tumour therapy.
An emerging wave of targeted therapeutic molecules against RTKs is composed of nucleic acid-based aptamers. They are short structured single-stranded RNA or DNA ligands that bind with high selectivity and sensitivity, due to their specific three-dimensional shapes, to their target molecules. Aptamers have a number of important advantages over proteins as therapeutic reagents (Cerchia et al., 2002; Cerchia and de Franciscis 2010). Indeed, they are entirely chemically synthesized by an in vitro evolution-based approach named SELEX (Systematic Evolution of Ligands by EXponential enrichment), thus avoiding the use of animal cells and assuring a rapid production process with high batch fidelity Furthermore, aptamers can be readily chemically modified by the addition of polyethylene glycol and other moieties to enhance their bioavailability and pharmacokinetics. Aptamers are non-immunogenic and, in addition, RNA made with pyrimidines modified at the 2′-position, which renders them resistant to extracellular nucleases, are even less immunogenic than natural RNA.
Li et al performed an in vitro selection against the purified extracellular domain of EGFR. The resulting RNA aptamer was then used to deliver gold nanoparticles to the intracellular compartment of cancer cells, offering proof-of-concept for further delivery strategies with this aptamer (Li et al., 2010). No functionality has been associated to the aptamer by Li et al. In the present invention a neutralizing RNA-aptamer specifically inhibiting the EGFR was identified.